Calcium/calmodulin-dependent phosphorylation of tumor protein D52 on serine residue 136 may be mediated by CAMK2δ6

Catherine S. Chew, Xunsheng Chen, Hanfang Zhang, Eric A. Berg, Han Zhang

Research output: Contribution to journalArticle

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Abstract

Tumor protein D52 is expressed at relatively high levels in cells within the gastrointestinal tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S136, that undergoes increased phosphorylation upon elevation of intracellular Ca2+ concentration. A phosphospecific antibody (pS136) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84, and HEK293 cells. By using D52 as a substrate, a protein kinase with a molecular weight (Mr) of ∼50 kDa was identified with "in gel" assays. This kinase comigrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2)α cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT286/287) antibody when activated. Carbachol-stimulated phosphorylation of S136 was inhibited by the CAMK2 inhibitor KN93 (IC50 38 μM) and by the calmodulin antagonist W7 (IC50 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK2δ6, which has the same domain structure and Mr as CAM2α, was identified in gastric mucosa by RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK2δ6 in the mediation of D52 phosphorylation.

Original languageEnglish (US)
Pages (from-to)G1159-G1172
JournalAmerican Journal of Physiology - Gastrointestinal and Liver Physiology
Volume295
Issue number6
DOIs
StatePublished - Dec 1 2008

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Keywords

  • HEK293 cells
  • Mouse gastric glands
  • Protein phosphorylation
  • T84 cells

ASJC Scopus subject areas

  • Physiology
  • Hepatology
  • Gastroenterology
  • Physiology (medical)

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